Mating-driven variability in olfactory local interneuron wiring

Variations in neuronal connectivity occur widely in nervous systems from invertebrates to mammals. Yet, it is unclear how neuronal variability originates, to what extent and at what time scales it exists, and what functional consequences it might carry. To assess inter- and intraindividual neuronal variability, it would be ideal to analyze the same identified neuron across different brain hemispheres and individuals. Here, using genetic labeling and electron microscopy connectomics, we show that an identified inhibitory olfactory local interneuron, TC-LN, exhibits extraordinary variability in its glomerular innervation patterns. Moreover, TC-LN’s innervation of the VL2a glomerulus, which processes food signals and modulates mating behavior, is sexually dimorphic, is influenced by female’s courtship experience, and correlates with food intake in mated females. Mating also affects output connectivity of TC-LN to specific local interneurons. We propose that mating-associated variability of TC-LNs regulates how food odor is interpreted by an inhibitory network to modulate feeding.

Glomeruli innervated by TC-LNs of virgin females and mated females The numbers of innervated glomeruli with innervation frequencies ≥ 1% are 21 and 24 in virgin (n = 823) and mated females (n = 1244) in y 1 w * background, respectively. Glomerulus DC1, VA5, VA7l are innervated by TC-LNs in mated females only. These glomeruli and their corresponding innervation frequencies are listed below.

GRASP signals of TC-LNs
The split GFPs were fused to CD4, which allow the spGFP to distribute along membranes of the expressed neurons. Accordingly, it has been a concern that a part of GRASP signals may be the result of contact between neurites, instead of the reconstructed GFP in the synapse. A single PN has most abundant dendrites in a given glomerulus. However, nearly 70%-80% analyzed ALs of TC-LN and GH146+ PN GRASP virgins or virgin males do not have GRASP signal (Fig. S13C). These results suggest the majority of GRASP signals uncovered in this study were originated from synapses.

trans-Tango
Based on the original design of the trans-Tango system, trans-Tango flies reared at 25 o C revealed less labeled postsynaptic cells than that in flies reared at 18 o C with comparable ages. In this study, flies were reared for either 7-day or 8-day before analyzing the signal, TC-LNs in these flies should have enough time to accumulate hGCG::hICAM1::dNRXN1 to trigger signals. Along this line, virgin females carrying TC-LNs tended to have no or weaker trans-Tango signals ( Fig. S13F and S13G), suggesting the synapses between TC LNs and their postsynaptic neurons may be much weak in virgins. The brains with scarce postsynaptic neurons also offer the near single cell type images that allow us to identify the morphologies and types of TC-LN postsynaptic neurons.
Possible recruited TC-LN synaptic neurons in mated females 449-QF labels ~60 lateral LNs that have positive GRASP signals with TC-LN, some of which were newly synapsed by TC-LN after mating. Brain myoinhibitory peptides (MIP) are expressed in brain tissue to inhibit food intake (64) and to enhance polyamide preference of mated females (65). The latter effect is mediated by binding of MIP to its receptor, sex peptide receptor (SPR). Interestingly, both MIP and SPR are expressed in subset(s) of lateral LNs (57,60). In the future, it will be interesting to test whether the lateral LNs newly synapsed to TC-LNs are Mip-and/or SPR-positive LNs. In addition, trans-Tango revealed at least one ventral LNs (1702323386) was a postsynaptic target of TC-LNs in mated females. This LN innervates a subset of glomeruli targeted by pheromone-sensing ORNs. It may form an additional inhibitory forward-loop by TC-LN in these pheromone-related glomeruli. , a single labeled second type of unilateral LN, pacman LN (yellow arrow, named after its innervation pattern, v2LN30) (E), and a bilateral LN (f, yellow arrow). Note that in (F), the contralateral antennal lobe has a labeled TC-LN (white arrow). Asterisks, non-LN neurons that innervate other neuropils than antennal lobes. Scale bars, 20 µm. (G to I) Two labeled cells in (A) belong to the following three categories: (1) one TC-LN (white arrow) and one pacman LN (yellow arrow) (G), (2) one TC-LN (white arrows) and one bilateral LN (yellow arrow; type 1) (H, right antennal lobe), and (3) one TC-LN (white arrow) and one bilateral LN (yellow arrow, type 2) (I). Asterisks, non-LN neurons that innervate other neuropils than antennal lobes. Scale bars, 20 µm.    Table S4). Note that DM6 showed anti-correlation with DA2, VL2a and Column. (C) The difference between the correlation of a given glomerular pair in males and females (Table S4). (D) Innervation frequencies of TC-LNs of male and female flies with the genetic background y 1 w * (left) and Canton S (right). Glomeruli with 100 % or 0 % innervation in both sexes were excluded. Chi-squared tests, followed by post hoc Bonferroni correction, were conducted to examine the differences between males and females in 43 glomeruli and 28 glomeruli in y 1 w * and Canton S, respectively (Table S3). *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S., not significant. N.D., not done; DA4l is not among the 28 glomeruli of Canton S. The Chi-squared tests may be overly sensitive in calling statistical difference in the y 1 w * group because of the very large numbers. (E) Variable glomerular innervations of TC-LNs caused by female courtship experience. The number of examined antennal lobes are 232, 139, and 310 TC-LNs in 0-2h-old virgins, 14-day-old mated females and 14-day-old virgins, respectively. Chi-squared test, followed by Bonferroni correction was conducted to examine the differences between virgins and mated females in 26 glomeruli in y 1 w * (Table S5, top). *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S, not significant. (F) VL2a innervations of TC-LNs caused by male courtship experience. The number of examined antennal lobes are 188, 69, and 46 TC-LNs in 0-2h-old virgin males, 14-day-old mated males and 14-day-old virgin males, respectively. Chisquared test, followed by Bonferroni correction was conducted to examine the differences between virgins and mated males in 26 glomeruli in y 1 w * (Table S5, bottom). Only the data of VL2a were shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S, not significant. in adult virgins, mated females, virgin males, and mated males. 15 randomly selected cells from each group were examined. See methods for a detailed description of the calculation. Kruskal-Wallis non-parametric one-way ANOVA was used to examine the differences of innervation densities (n = 60) across glomeruli. p = 1.445e-80. *, p < 0.05; **, p < 0.01; ***, p < 0.001; N.S, not significant. (B) Process densities of single TC-LNs as in (A) were grouped according to the binary innervation score of the corresponding cells. 1, innervated; 0, not innervated. Note that the density profile lost the power to distinguish true innervation when sparse processes innervated a particular glomerulus (e.g., DL1 and VL2a).

Fig. S8. Sexually dimorphic VL2a innervation by TC-LNs is driven by courtship experience in females.
(A-C) Removing sperm (A), sex peptide (B) and cuticular hydrocarbons (C) from males did not affect courtship-driven TC-LN innervation in VL2a. bol mutant and oenocyte (OE) males fail to produce sperm and cuticular hydrocarbons, respectively. Parentheses show the total number of cells examined. Chi-squared test, followed by Bonferroni correction was used to examine the differences between pairs. N.S., not significant.  . Three-way ANOVA (A-D) was used to examine the main effects and interactions of variables, followed by post hoc Bonferroni test to analyze multiple comparisons among different conditions. *p < 0.05, **p < 0.01, ***p < 0.001.     The trans-Tango system includes three major components, hGCGR::TEVcs::QF (pink), hArr::TEV (magenta) and hGCG::hICAM1::dNRXN1 (blue). The first two components are ubiquitously expressed in all cells. By contrast, the ligand hGCG::hICAM1::dNRXN1 is driven by GAL4. When no GAL4 is expressed in the pre-synapse (left), the trans-Tango system is off. When GAL4 is expressed in the presynaptic neurons, it drives the expression of mCD8GFP (green) and hGCG:hICAM1:dNRXN1. The ligand will bind to postsynaptic receptors hGCG:TEVcs:QF (right), which then recruit hArr::TEV to its cytosolic tail, causing the cleavage and release of QF.  Table S9.     Figure S4B and differences between the correlation of glomerular pairs of TC-LNs in males and females in Figure S4C.    Figures 4G, S7D, S8A-C and S13C.      Three-way ANOVA was used to examine the main effects and interactions of variables and the post hoc Bonferroni correction was used to analyze multiple comparisons among different conditions. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Table S9. Genotypes of flies used in experiments described in Figures 1-4 and S1-S13.   S1D, S1E, S1F, S1G, S1H